RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Orlando, Tereza Cristina; Mayer, Mario Gustavo; Campbell, David A.; Sturm, Nancy R.; Flöeter-Winter, Lucile Maria

doi:10.1590/s0074-02762007005000123

Cited by 9 publications

(7 citation statements)

References 25 publications

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“…Several authors reported successful labeling of recombinant proteins in methylotrophic yeast [38,39], instead a limited number of studies reported successful isotopic labeling in baculoviral expression system [40,41]. Niculae et al [42] and Foldynovà-Trantirkovà et al [43] reported a NMR analysis of Enhanced Green Fluorescent Protein (EGFP) purified from recombinant L. tarentolae strain cultivated in a synthetic medium for selective 15 N- Chloramphenicol acetyl transferase Intracellular --- [45] Cu/Zn Superoxide dismutase Intracellular --30 [8,9] G-protein coupled receptor 56 (Ex segment) Secreted Native -0.1-5 [8] Erythropoietin Secreted Native -30 [8,9] Erythropoietin Secreted Acid phosphatase -0. T7 RNA polymerase Intracellular --30 [8,9] Tissue plasminogen activator Intracellular --0.17 [52] WASP Intracellular --0.1-5 [8] labeling.…”

Section: Expression Of Proteins For Structural Studiesmentioning

confidence: 99%

Recombinant Protein Expression in Leishmania tarentolae

Basile

Peticca²

2009

Mol Biotechnol

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A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals, and plants. Recently Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has been suggested as candidate for heterologous genes expression. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein; the oligosaccharide structures are similar to those of mammals with N-linked galactose, and fucose residues. To date several heterologous proteins have been expressed in L. tarentolae including both cytoplasmic enzymes and membrane receptors. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of those tools coupled with a better understanding of the biology of Leishmania species will lead to value and power in commercial and research labs alike.

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Section: Expression Of Proteins For Structural Studiesmentioning

confidence: 99%

Recombinant Protein Expression in Leishmania tarentolae

Basile

Peticca²

2009

Mol Biotechnol

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“…Therefore, the triple fusion gene was integrated into the rRNA locus downstream of 18S ribosomal promoter [19] to be transcribed by RNA Pol I, or expressed episomally under regulation signals or regulatory elements of RNA Pol I. However, in both systems, exogenous protein was expressed from the strong rRNA promoter and transcribed by the endogenous RNA Pol I [18,28]. The specific sequences of ribosomal rRNA promoter in plasmid vector allowed replication and its stable maintenance during inheritance as a single copy [29].…”

Section: Discussionmentioning

confidence: 99%

Expressional Comparison Between Episomal and Stable Transfection of a Selected tri-fusion Protein in Leishmania tarentolae

Taheri

Gholami

Saatchi

et al. 2014

vacres

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Introduction: Leishmania tarentolae (L. tarentolae) is a nonpathogenic species of Leishmania genus that can be used as an expression system to produce immunogenic proteins or epitopes in vivo acting as an efficient and safe recombinant live vector in vaccinology. Cysteine proteases (CPs) are one group of Family C1 peptidases in Leishmania that are important for survival of the parasite within the host and are introduced as potential vaccine candidates. Two types of cysteine proteases, CPA and CPB, play a critical role in Leishmania. Previously, it has indicated that immunization with two genes or recombinant proteins of CPA and CPB individually or fused together with various adjuvant or combined with liposome are able to elicit a protective immune response against L. major in BALB/c mice. Methods: In this study, for the first time, two lines of recombinant non-pathogenic Leishmania were generated by transfection of a heterologous triple fusion gene encoding cpa/cpb/egfp. For this purpose, two different expressions approaches were taken; episomal expression using rDNA promoter, and integrative expression from rRNA locus of genome. Results: After genotype confirmation, the fluorescence intensity was monitored in different phases of parasite growth by both fluorescence microscopy and FACS analysis.Western blot and RT-PCR analysis showed that cpa/cpb/egfp tri-fused genes were specifically expressed in both lines of transgenic parasites. Conclusion: In this study a single fused gene was expressed in both systems and the results showed that the expression of integrated gene was higher than episomal. In addition, the present data suggest that L. tarentolae expressing cpa/cpb/egfp is a promising carrier as vectored vaccine in future research. Vac Res, 2014, 1 (1) : 1 -9

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“…The recent sequencing of the Leishmania spp. genomes indicates that protein-coding genes are organized as polycistronic units (Ivens et al 2005;Clayton and Shapira 2007;Haile and Papadopoulou 2007;Orlando et al 2007). Transcription in this organism initiates at strand the switch regions on each chromosome, without defined RNA polymerase II promoters or other typical transcription factors (Martinez-Calvillo et al 2003).…”

Section: Discussionmentioning

confidence: 99%

Cloning and expression of human IFN-γ in Leishmania tarentolae

Davoudi

Azam

Khodayari

et al. 2011

World J Microbiol Biotechnol

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Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-c), an antiviral pro-inflammatory cytokine, has broadened interest in optimizing methods for its production. We herein describe a unicellular eukaryotic system, Leishmania tarentolae, a Trypanosomatidae protozoan parasite of gecko Tarentola annularis, which has recently been introduced as a candidate for heterologous gene expression. In this study, the hIFN-c cDNA was amplified from phyto-hemagglutininstimulated peripheral blood mononuclear cells of a healthy blood donor using RT-PCR. In order to express, the rhIFNc protein, the resulting cDNA was cloned in two expression cassettes (each containing one copy of hIFN-c cDNA) and integrated into the small subunit of ribosomal RNA gene of L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Western blotting of rhIFN-c and ELISA confirmed the expression and production of 9.5 mg of rhIFN-c protein/l respectively.

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RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Cited by 9 publications

References 25 publications

Recombinant Protein Expression in Leishmania tarentolae

Recombinant Protein Expression in Leishmania tarentolae

Expressional Comparison Between Episomal and Stable Transfection of a Selected tri-fusion Protein in Leishmania tarentolae

Cloning and expression of human IFN-γ in Leishmania tarentolae

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